Centrioles are the duplicating elements of centrosomes, organelles which nucleate microtubule growth to build mitotic/meiotic spindles and cilia. During each normal cell division, centrioles duplicate once and once only. Like DNA replication, centriole duplication is a semi-conservative process tightly coupled to the cell cycle and occurs only during S-phase. Centriole number must be closely regulated because a deficiency or an excess of centrioles may interfere with correct chromosome segregation and asymmetric stem cell division. The presence of too few centrioles contributes to various ciliopathies, including microcephaly, and Seckel syndrome. In contrast, too many centrioles directly promotes chromosomal instability (CIN) a driving force for aneuploidy that induces miscarriage, birth defects, and tumorigenesis. Furthermore, the presence of too many centrioles is frequently observed in cancer cells. Normally, each `mother' centriole assembles only a single `daughter', but centrioles have the capacity to assemble multiple daughters simultaneously - an aneuploidy- generating condition called centriole amplification. Normally, only a single, restricte site on the mother centriole is used to spawn a daughter. However, the mechanism limiting assembly to only this site is unknown. Experiments in this proposal test a hypothesis that explains why centriole duplication is normally restricted to a single event per mother centriole. Specifically, we link three conserved centriolar proteins, Polo-like kinase 4 (Plk4), its binding-partner and scaffolding protein Asterless (Asl), and the centriole assembly protein Anastral Spindle 2 (Ana2) into a single regulatory pathway. Overexpression of any of these proteins induces assembly of multiple daughter centrioles. How the cell cycle control program regulates these key proteins to limit daughter centriole assembly to only a single event is unknown. Here, we hypothesize that cell cycle regulators, including the Anaphase Promoting Complex (APC) ubiquitin-ligase, control the destruction of a Plk4/Asl/Ana2 complex to alter their pattern/activit on mother centrioles and generate a single site for daughter assembly. Our preliminary data indicate that 1) the Plk4 pattern on centrioles changes during mitotic progression: first coating the entire mother centriole but then being pruned to a single asymmetric spot by mitotic exit; 2) Plk4 and Ana2 are APC targets; 3) phosphorylation protects subpopulations of these proteins from degradation; and 4) Asl is a Plk4 substrate that stabilizes mitotic Plk4. In this proposal, we will (1) determine if APC regulation of Plk4 localization on centrioles defines the site of daughte centriole assembly, (2) determine the functional interaction between Plk4 and Asl which assists in defining a single site of centriole assembly, and (3) determine if Ana2 regulates Plk4 activation and whether Ana2 is controlled by APC. These studies will provide mechanistic insight into a molecular process that governs the fidelity of centriole duplication. Understanding the process at the molecular level will guide future studies to explore the etiology of centriole amplification during tumorigenesis and other centriole-related diseases.